The overall goal of this proposal is to define the molecular signaling mechanisms crucial for megakaryopoiesis. The production and activation of platelets, the cells which mediate primary hemostasis, are precisely regulated over the life of an organism through pathways that are not currently understood. The c-Mpl proto-oncogene, an orphan cytokine receptor, became the focus of intense investigation after it was shown that it is expressed almost exclusively on megakaryocytes, their precursors and progeny, and that antisense oligonucleotides specifically inhibited megakaryocyte colony formation from selected bone marrow cells. Subsequently, the Mpl-ligand was cloned and found to induce megakaryocyte proliferation and differentiation. These events now make it possible to study the intracellular events triggered by c-Mpl and its ligand, termed thrombopoietin. In previous studies we have shown that thrombopoietin induces the assembly of membrane and cytoplasmic protein complexes and the rapid tyrosine phosphorylaiton of many cytoplasmic proteins, including members of th Janus family of tyrosine kinases and the STAT family of latent transcriptional activators. Assessment of the roles of each of these proteins in megakaryocyte proliferation and differentiation should help identify those processes unique to megakaryopoiesis. Specific aims for this application are; 1) Define domains within the cytoplasmic portion of c-Mpl required for proliferation. 2) Characterize megakaryocyte differentiation in order to test the role of c-Mpl and its subdomains. 3) Investigate the function of JAK2 and TYK2 in thrombopoietin signaling. 4) Study the importance of PI-3 kinase and its physical association with c-Mpl. 5) Determine which STATs are activated by thrombopoietin and clone novel megakaryocyte STATs In addition to providing a more complete understanding of the biology of thrombopoietin, a critical regulator of platelet production, these studies may have significant implications for medical research. It is likely that many human diseases are caused by spontaneous mutation of signal transduction molecules. Essential thrombocytosis, myelodysplasia, and megakaryocyte leukemia are examples of diseases in which a constitutively active thrombopoietin signaling pathway may play a role. Amegakaryocytic thrombocytopenia and aplastic anemia may result from inactivating mutations. A better understanding of the mechanisms which underlie megakaryocytic development is a necessary prerequisite to providing new therapeutic options for patients with hematopoietic disorders.